Zhang Shengye, Wotzkow Carlos, Bongoni Anjan K, Shaw-Boden Jane, Siegrist Mark, Taddeo Adriano, Blank Fabian, Hofstetter Willy, Rieben Robert
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
Department of Clinical Research, University of Bern, Bern, Switzerland.
Bone. 2017 Apr;97:278-286. doi: 10.1016/j.bone.2016.12.007. Epub 2017 Jan 31.
Ischemia/reperfusion (I/R) injury has been extensively studied in organs such as heart, brain, liver, kidney, and lung. As a vascularized organ, bone is known to be susceptible to I/R injury too, but the respective mechanisms are not well understood to date. We therefore hypothesized that, similar to other organs, plasma cascade-induced inflammation also plays a role in bone I/R injury. Reperfusion injury in rat tibia was induced by unilateral clamping of the femoral artery and additional use of a tourniquet, while keeping the femoral vein patent to prevent venous congestion. Rats were subjected to 4h ischemia and 24h reperfusion. Deposition of complement fragment C3b/c and fibrin as well as expression of tissue factor (TF), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and E-selectin was detected by immunohistochemistry. In plasma, the levels of high mobility group box1 (HMGB1) were measured by ELISA. The total level of complement in serum was assessed by the CH50 test. Our results show that deposition of C3b/c was significantly increased with respect to healthy controls in cortical bone as well as in marrow of reperfused limbs. C3b/c deposition was also increased in cortical bone, but not in bone marrow, of contralateral limbs. Deposition of fibrin, as well as expression of PAI-1, was significantly increased in bone after ischemia and reperfusion, whereas expression of tPA was reduced. These differences were most prominent in vessels of bone, both in marrow and cortical bone, and both in reperfused and contralateral limbs. However, PAI-1, was only increased in vessels of reperfused cortical bone and there were no significant changes in expression of E-selectin. With respect to solid bone tissue, a significant increase of C3b/c and fibrin deposition was shown in osteocytes, and for fibrin also in the bone matrix, in both contralateral and reperfused cortical bone compared with normal healthy controls. A slight expression of TF was visible in osteocytes of the normal healthy control group, while TF was not present in the experimental groups. Moreover, CH50 values in serum decreased over time and HMGB1 was significantly increased in plasma of animals at the end of reperfusion. We conclude that ischemia and reperfusion of bone leads to activation of the complement and coagulation systems and a downregulation of the fibrinolytic cascade. In the acute phase, a vascular inflammation induced by activation of the plasma cascade systems also occurs in the bone. This is similar to I/R injury of other vascularized organs and tissues.
缺血/再灌注（I/R）损伤已在心脏、脑、肝、肾和肺等器官中得到广泛研究。作为一个血管化器官，骨骼也已知易受I/R损伤影响，但其具体机制迄今尚未完全明确。因此，我们推测，与其他器官类似，血浆级联反应诱导的炎症在骨骼I/R损伤中也发挥作用。通过单侧夹闭股动脉并额外使用止血带来诱导大鼠胫骨的再灌注损伤，同时保持股静脉通畅以防止静脉淤血。大鼠经历4小时缺血和24小时再灌注。通过免疫组织化学检测补体片段C3b/c和纤维蛋白的沉积以及组织因子（TF）、组织纤溶酶原激活物（tPA）、纤溶酶原激活物抑制剂-1（PAI-1）和E-选择素的表达。在血浆中，通过酶联免疫吸附测定法（ELISA）测量高迁移率族蛋白B1（HMGB1）的水平。通过50%溶血补体（CH50）试验评估血清中的总补体水平。我们的结果表明，与健康对照组相比，再灌注肢体的皮质骨和骨髓中C3b/c的沉积显著增加。对侧肢体的皮质骨中C3b/c沉积也增加，但骨髓中未增加。缺血和再灌注后，骨骼中纤维蛋白的沉积以及PAI-1的表达显著增加，而tPA的表达降低。这些差异在骨髓和皮质骨的骨血管中最为明显，在再灌注肢体和对侧肢体中均如此。然而，PAI-1仅在再灌注皮质骨的血管中增加，E-选择素的表达无显著变化。对于实体骨组织，与正常健康对照组相比，对侧和再灌注皮质骨的骨细胞中C3b/c和纤维蛋白沉积显著增加，纤维蛋白在骨基质中也增加。正常健康对照组的骨细胞中可见轻微的TF表达，而实验组中未检测到TF。此外，血清中的CH50值随时间下降，再灌注结束时动物血浆中的HMGB1显著增加。我们得出结论，骨骼的缺血和再灌注导致补体和凝血系统激活以及纤维蛋白溶解级联反应下调。在急性期，血浆级联系统激活诱导的血管炎症也发生在骨骼中。这与其他血管化器官和组织的I/R损伤相似。
